1. Field of the Invention
The present invention relates generally to the fields of allergy, immunology and cell biology. More specifically, the present invention relates to novel inhibitors of glucose-6-phosphate uptake and methods of using such glucose-6-phosphate uptake inhibitors to treat specific diseases.
2. Description of the Related Art
The inositol (1,4,5)tris-phosphate (IP.sub.3)-mediated release of calcium sequestered within the endoplasmic reticulum into the cytoplasm is an early step in a host of signal transduction cascades, including those initiated by ligation of antigen receptors in cells of the immune system. In unstimulated cells, calcium is sequestered within the endoplasmic reticulum to concentrations on the order of 10 mM through the action of calcium ATPases found in the membranes of the endoplasmic reticulum or its analogue in muscle, the sarcoplasmic reticulum. In addition to this pump, a mechanism for "buffering" intra-organellar calcium is also essential, since a free intra-organellar calcium concentration of 1 mM inhibits further sequestration. High-capacity, low-affinity calcium-binding proteins like calsequestrin or calreticulin within the endoplasmic reticulum have been proposed to be necessary to decrease the concentration of intra-organellar free calcium and allow the ATPase to continue to function. However, Volpe et al. determined that 3.6 mM calcium within the sarcoplasmic reticulum of frog muscle is not associated with calsequestrin and about 75% of the calcium released in response to excitation is not from a calsequestrin-associated pool.
In addition, isolated vesicles derived from the sarcoplasmic reticulum of muscle or the endoplasmic reticulum of other cells exhibit a relatively low calcium accumulating capacity. This capacity can be enhanced to physiological levels through the use of selected anions. In early experiments, high concentrations of Pi or oxalate were used to precipitate calcium and thus allow continued activity of the calcium ATPase. The possibility that anions might be physiologically relevant was first suggested by Chu et al. They found, using high concentrations of succinate, a significantly increased uptake of calcium by sarcoplasmic reticulum vesicles that was matched by the equimolar uptake of the negatively charged succinate, without precipitation. A stoichiometric efflux of the counter-ion was induced when calcium was released with an ionophore. Fulceri and co-workers found in microsomes from both liver and other tissues that physiological levels of P.sub.i also support an enhancement of calcium sequestration. Again, the calcium and the P.sub.i remained soluble, were imported in an equimolar ratio, and were rapidly released stoichiometrically upon treatment with calcium ionophore, IP.sub.3, or dilution of either extramicrosomal calcium or P.sub.i.
These data provide support for the idea that anions, by forming a soluble complex with calcium, are able to restore the calcium-sequestering capacity of the endoplasmic reticulum to physiological levels and in so doing provide an enlarged pool of calcium for release in response to stimuli. If an anion that is used as a "buffer" for intraorganellar calcium is also imported and exported along with calcium, the need for a compensating current to balance the movement of calcium's charge during both the transport and release processes is also met. However, the relevance of these observations to the physiology of intact cells and the identity of the anion imported into the endoplasmic reticulum, if any, remains undetermined.
An additional anion has been implicated in the enhancement of endoplasmic reticulum calcium stores. Glucose-6-phosphate (G-6-P) is efficiently imported into the endoplasmic reticulum of liver as a step in glycogenolysis. It is cleaved to glucose and P.sub.i by an intra-organellar glucose-6-phosphatase, and the products are then re-exported to the cytoplasm. Benedetti et al. showed that in liver microsomes less than 1 mM glucose-6-phosphate resulted in a 10-fold increase in sequered calcium, while P.sub.i as a counter-ion was much less effective. Similar results were found with microsomes from kidney and pancreatic cells, both of which are characterized by high levels of glucose-6-phosphatase.
Although immune cells have not been reported to possess high levels of glucose-6-phosphatase, glucose analogues have been shown to effect calcium-mediated immune responses; T cell-mediated cytolysis, IgG-mediated phagocytosis by macrophages and neutrophils, and antibody-dependent eosinophil-mediated lysis of schistosomula have been known for many years to be inhibited by the glucose analogue 2-deoxyglucose. Since 2-deoxyglucose leads to a decrease in cellular ATP, its inhibitory effects were not unexpected. However, closer examinations demonstrated that the inhibitory effect was not due to ATP depletion nor to 2-deoxyglucose's inhibitory effect on glycoprotein synthesis. In addition, another 2-carbon derivative of glucose, glucosamine has been shown to act as an anti-reactive and anti-inflammatory and has been used to treat osteoarthritis although the mechanism underlying its efficacy is undetermined.
Other calcium related phenomena as diverse as smooth muscle contractility in response to norepinephrine or pressor hormones, sperm capacitation, and neural transmission both in vivo and in vitro have also been reported to have requirements for glucose independent of total ATP levels. In the case of smooth muscle Zhang and Paul reported that the absence of glucose led to higher than normal cytoplasmic calcium due to ineffective calcium sequestration by the endoplasmic reticulum/sarcoplasmic reticulum. The dependency on glucose was suggested to be due to the presence of an ATP pool generated by glycolysis that is independent of total cellular ATP and necessary to achieve proper calcium sequestration within the endoplasmic reticulum/sarcoplasmic reticulum.
The prior art continues to lack of additional effective means of treating various auto-immune diseases. The present invention fulfills this longstanding need and desire in the art.